Seed-preferred promoters from end genes

ABSTRACT

The invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions are novel nucleotide sequences for seed-preferred promoters isolated from genes for end1 and end2. A method for expressing a heterologous nucleotide sequence in a plant using the disclosed promoter sequences is provided. The method comprises transforming a plant cell to comprise a heterologous nucleotide sequence operably linked to one of the promoters of the invention and regenerating a stably transformed plant from the transformed plant cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of co-pending application U.S.Ser. No. 09/383,543 filed Aug. 26, 1999, which application claims thebenefit of U.S. application Ser. No. 60/098,230 filed Aug. 28, 1998, nowabandoned, which is herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of plant molecularbiology, more particularly to regulation of gene expression in plants.

BACKGROUND OF THE INVENTION

[0003] Expression of heterologous DNA sequences in a plant host isdependent upon the presence of an operably linked promoter that isfunctional within the plant host. Choice of the promoter sequence willdetermine when and where within the organism the heterologous DNAsequence is expressed. Where continuous expression is desired throughoutthe cells of a plant, constitutive promoters are utilized. In contrast,where gene expression in response to a stimulus is desired, induciblepromoters are the regulatory element of choice. Where expression inspecific tissues or organs are desired, tissue-specific promoters may beused. That is, they may drive expression in specific tissues or organs.Such tissue-specific promoters may be constitutive or inducible. Ineither case, additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

[0004] Frequently it is desirable to have constitutive or inducibleexpression of a DNA sequence in particular tissues or organs of a plant.For example, increased nutritional value of a plant might beaccomplished by genetic manipulation of the plant's genome to comprise aseed-preferred promoter operably linked to a heterologous gene such thatproteins with enhanced amino acid content are produced in the seed ofthe plant.

[0005] Alternatively, it might be desirable to inhibit expression of anative DNA sequence within a plant's tissues to achieve a desiredphenotype. In this case, such inhibition might be accomplished withtransformation of the plant to comprise a tissue-specific promoteroperably linked to an antisense nucleotide sequence, such thatconstitutive expression of the antisense sequence produces an RNAtranscript that interferes with translation of the mRNA of the nativeDNA sequence.

[0006] Seed development involves embryogenesis and maturation events aswell as physiological adaptation processes that occur within the seed toinsure progeny survival. Developing plant seeds accumulate and storecarbohydrate, lipid, and protein that are subsequently used duringgermination. Expression of storage protein genes in seeds occursprimarily in the embryonic axis and cotyledons and in the endosperm ofdeveloping seeds but rarely in mature vegetative tissues. Generally, theexpression patterns of seed proteins are highly regulated. Thisregulation includes spatial and temporal regulation during seeddevelopment. A variety of proteins accumulate and decay duringembryogenesis and seed development and provide an excellent system forinvestigating different aspects of gene regulation as well as forproviding regulatory sequences for use in genetic manipulation ofplants.

[0007] Thus, isolation and characterization of seed-preferred promotersthat can serve as regulatory regions for expression of heterologousnucleotide sequences of interest in a seed-preferred manner are neededfor genetic manipulation of plants.

SUMMARY OF THE INVENTION

[0008] It is an object of the present invention to provide a novelnucleotide sequence for modulating gene expression in a plant.

[0009] It is a further object of the present invention to provide anisolated promoter capable of driving transcription in a seed-preferredmanner.

[0010] It is a further object of the present invention to provide amethod of improved control of an endogenous or exogenous product in theseed of a transformed plant.

[0011] It is a further object of the present invention to provide amethod for providing useful changes in the phenotype of a seed of atransformed plant.

[0012] It is a further object of the present invention to provide amethod for producing a novel product in the seed of a transformed plant.

[0013] It is a further object of the present invention to provide amethod for producing a novel function in the seed of a transformedplant.

[0014] Therefore, in one aspect, the present invention relates to anisolated nucleic acid comprising an isolated promoter that is capable ofdriving transcription in a seed-preferred manner, wherein the promotercomprises a nucleotide sequence selected from the group consisting of:

[0015] a) a sequence comprising a fragment of the nucleotide sequenceset forth in SEQ ID NO:4; and

[0016] b) the nucleotide sequence set forth in SEQ ID NO:4.

[0017] In other aspects, the present invention relates to expressioncassettes comprising the promoter operably linked to a nucleotidesequence, vectors containing the expression cassette, and plants stablytransformed with at least one expression cassette.

[0018] In a further aspect, the present invention relates to a methodfor modulating expression in the seed of a stably transformed plantcomprising the steps of (a) transforming a plant cell with an expressioncassette comprising the promoter of the present invention operablylinked to at least one nucleotide sequence; (b) growing the plant cellunder plant growing conditions and (c) regenerating a stably transformedplant from the plant cell wherein expression of the nucleotide sequencealters the phenotype of the seed.

[0019] Compositions and methods for regulating expression ofheterologous nucleotide sequences in a plant are provided. Compositionsare novel nucleotide sequences for seed-preferred plant promoters, moreparticularly transcriptional initiation regions isolated from the plantgenes end1 and end2. A method for expressing a heterologous nucleotidesequence in a plant using the transcriptional initiation sequencesdisclosed herein is provided. The method comprises transforming a plantcell with a transformation vector that comprises a heterologousnucleotide sequence operably linked to one of the plant promoters of thepresent invention and regenerating a stably transformed plant from thetransformed plant cell. In this manner, the promoter sequences areuseful for controlling the expression of endogenous as well as exogenousproducts in a seed-preferred manner.

[0020] Downstream from and under the transcriptional initiationregulation of the seed-specific region will be a sequence of interestwhich will provide for modification of the phenotype of the seed. Suchmodification includes modulating the production of an endogenousproduct, as to amount, relative distribution, or the like, or productionof an exogenous expression product to provide for a novel function orproduct in the seed.

DETAILED DESCRIPTION OF THE INVENTION

[0021] In accordance with the invention nucleotide constructs areprovided that allow initiation of transcription in seed. Constructs ofthe invention comprise regulated transcriptional initiation regionsassociated with seed formation and seed tissues. Thus, the compositionsof the present invention comprise novel nucleotide sequences for plantpromoters, particularly seed-preferred promoters, more particularlyendosperm specific promoters, for the genes end1 and end2. The end1promoter drives expression in transfer cells at an early stage inprecursor cells and continues expression into mature cells. The end2promoter drives expression in aleurone cells.

[0022] The promoters for these genes may be isolated from the 5′untranslated region flanking their respective transcription initiationsites. Methods for isolation of promoter regions are well known in theart.

[0023] The term “isolated” refers to material, such as a nucleic acid,which is: (1) substantially or essentially free from components whichnormally accompany or interact with it as found in its naturallyoccurring environment; the isolated material optionally comprisesmaterial not found with the material in its natural environment; or (2)if the material is in its natural environment, the material has beensynthetically (non-naturally) altered or produced by deliberate humanintervention to a composition and/or placed at a locus in the cell(e.g., genome or subcellular organelle) not native to a material foundin that environment. The alteration to yield the synthetic material canbe performed on the material within or removed from its natural state.

[0024] Methods are readily available in the art for the hybridization ofnucleic acid sequences. Promoter sequences from other plants may beisolated according to well-known techniques based on their sequencehomology to the promoter sequences set forth herein. In thesetechniques, all or part of the known promoter sequence is used as aprobe which selectively hybridizes to other sequences present in apopulation of cloned genomic DNA fragments (i.e. genomic libraries) froma chosen organism.

[0025] For example, the entire promoter sequence or portions thereof maybe used as probes capable of specifically hybridizing to correspondingpromoter sequences. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length, and most preferablyat least about 20 nucleotides in length. Such probes may be used toamplify corresponding promoter sequences from a chosen organism by thewell-known process of polymerase chain reaction (PCR). This techniquemay be used to isolate additional promoter sequences from a desiredorganism or as a diagnostic assay to determine the presence of thepromoter sequence in an organism.

[0026] Such techniques include hybridization screening of plated DNAlibraries (either plaques or colonies; see e.g. Innis et al. (1990) PCRProtocols, A Guide to Methods and Applications, eds., Academic Press).

[0027] The terms “stringent conditions” or “stringent hybridizationconditions” includes reference to conditions under which a probe willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

[0028] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

[0029] For purposes of defining the invention preferably low stringencyconditions are employed including hybridization with a buffer solutionof 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50 to 55° C. More preferably moderate stringency conditionsare employed including hybridization in 40 to 45% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Mostpreferably high stringency conditions are employed includinghybridization in 50% formamide, 1 M NaCl, 1 % SDS at 37° C., and a washin 0.1×SSC at 60 to 65° C. Hybridization times are not critical and canrange from about four hours to about sixteen hours.

[0030] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-lnterscience, N.Y. (1995). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.) In general, sequences thatcorrespond to promoter sequences of the invention and hybridize to thepromoter sequence disclosed herein will be at least 50% homologous,preferably 60%, 70%, 80%, 85%, 90%, and even 95% homologous or more withthe disclosed sequences.

[0031] The promoter regions of the invention may be isolated from anyplant, including, but not limited to corn (Zea mays), (Brassica napus,Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), oats, barley,vegetables, ornamentals, and conifers. Preferably plants include corn,soybean, sunflower, safflower, oil seed Brassica, wheat, rice, barley,rye, alfalfa, and sorghum.

[0032] The coding sequence expressed by the promoters of the inventionmay be used for varying the phenotype of the seeds. Various changes inphenotype are of interest including modifying the fatty acid compositionin seeds, altering the starch or carbohydrate profile, altering theamino acid content of the seed, and the like. These results can beachieved by providing expression of heterologous or increased expressionof endogenous products in seeds. Alternatively, the results can beachieved by providing for a reduction of expression of one or moreendogenous products, particularly enzymes or cofactors in the seed.These changes result in a change in phenotype of the transformed seed.

[0033] Genes of interest include, generally, those involved in oil,starch, protein, carbohydrate or nutrient metabolism as well as thoseaffecting kernel size, sucrose loading, and the like. In particular,end1 may find use in regulating the influx of nutrients. Both promotersare useful in disease resistance and in regulating expression of phytategenes particularly to lower phytate levels in the seed.

[0034] General categories of genes of interest for the purpose ofpresent invention include for example, those genes involved ininformation, such as Zinc fingers, those involved in communication, suchas kinases, and those involved in housekeeping, such as heat shockproteins. More specific categories of transgenes, for example, includegenes encoding important traits for agronomics, insect resistance,disease resistance, herbicide resistance, and grain characteristics. Itis recognized that any gene of interest can be operably linked to thepromoter of the invention and expressed in the seed.

[0035] Important traits such as oil, starch and protein content can begenetically altered in addition to using traditional breeding methods.Modifications include altering the content of oleic acid, saturated andunsaturated oils, increasing levels of lysine and sulfur-containingamino acids and providing other essential amino acids, and alsomodification of starch. Hordothionin protein modifications are describedin WO94/16078; WO96/38562; WO96/08220; and U.S. Pat. No. 5,703,409issued Dec. 30, 1997, the disclosures of which are incorporated hereinin their entirety by reference. Another example is lysine and/or sulfurrich seed protein encoded by the soybean 2S albumin described inWO97/35023, and the chymotrypsin inhibitor from barley, Williamson etal. (1987) Eur. J Biochem. 165:99-106, the disclosures of each areincorporated by reference. Derivatives of the following genes can bemade by site directed mutagenesis to increase the level of preselectedamino acids in the encoded polypeptide. For example, the gene encodingthe barley high lysine polypeptide (BHL), is derived from barleychymotrypsin inhibitor, WO98/20133, incorporated herein by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley et al. (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs;Applewhite, H. (ed.); American Oil Chemists Soc., Champaign, Ill.,497-502, incorporated herein in its entirety by reference), corn(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359, both incorporated herein in its entirety by reference) andrice (Musumura et al. (1989) Plant Mol. Biol. 12:123, incorporatedherein in its entirety by reference). Other agronomically importantgenes encode latex, Floury 2, growth factors, seed storage factors andtranscription factors.

[0036] The quality of grain is reflected in traits such as levels andtypes of oils, saturated and unsaturated, quality and quantity ofessential amino acids, and levels of cellulose. In corn, modifiedhordothionin proteins, described in WO94/16078; WO96/38562; WO96/08220;and U.S. Pat. No. 5,703,409; provide descriptions of modifications ofproteins for desired purposes.

[0037] Commercial traits can also be encoded on a gene(s) which couldalter or increase for example, starch for the production of paper,textiles, and ethanol, or provide expression of proteins with othercommercial uses. Another important commercial use of transformed plantsis the production of polymers and bioplastics such as described in U.S.Pat. No. 5,602,321 issued Feb. 11, 1997. Genes such as B-ketothiolase,PHBase (polyhydroxyburyrate synthase) and acetoacetyl-CoA reductase (seeSchubert et al. (1988) J. Bacteriol 170(12):5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

[0038] Exogenous products include plant enzymes and products as well asthose from other sources including prokaryotes and other eukaryotes.Such products include enzymes, cofactors, hormones, and the like. Thelevel of seed proteins, particularly modified seed proteins havingimproved amino acid distribution to improve the nutrient value of theseed can be increased. This is achieved by the expression of suchproteins having enhanced amino acid content.

[0039] Insect resistance genes may encode resistance to pests that havegreat yield drag such as rootworm, cutworm, European Corn Borer, and thelike. Such genes include, for example, Bacillus thuringiensis endotoxingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; Geiseret al. (1986) Gene 48:109); lectins (Van Damme et al.(1994) Plant Mol. Biol. 24:825); and the like.

[0040] Genes encoding disease resistance traits may includedetoxification genes, such as against fumonosin (U.S. patent applicationSer. No. 08/484,815 filed Jun. 7, 1995); avirulence (avr) and diseaseresistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al.(1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089; and thelike.

[0041] Agronomic traits in seeds can be improved by altering expressionof genes that affect the response of seed growth and development duringenvironmental stress, Cheikh-N et al (1994) Plant Physiol. 106(1):45-51)and genes controlling carbohydrate metabolism to reduce kernel abortionin maize, Zinselmeier et al. (1995) Plant Physiol. 107(2):385-391.

[0042] As noted, the heterologous nucleotide sequence operably linked toone of the promoters disclosed herein may be an antisense sequence for atargeted gene. By “antisense DNA nucleotide sequence” is intended asequence that is in inverse orientation to the 5′-to-3′ normalorientation of that nucleotide sequence. When delivered into a plantcell, expression of the antisense DNA sequence prevents normalexpression of the DNA nucleotide sequence for the targeted gene. Theantisense nucleotide sequence encodes an RNA transcript that iscomplementary to and capable of hybridizing to the endogenous messengerRNA (mRNA) produced by transcription of the DNA nucleotide sequence forthe targeted gene. In this case, production of the native proteinencoded by the targeted gene is inhibited to achieve a desiredphenotypic response. Thus the promoter sequences disclosed herein may beoperably linked to antisense DNA sequences to reduce or inhibitexpression of a native protein in the plant seed.

[0043] By “promoter” or “transcriptional initiation region” is intendeda regulatory region of DNA usually comprising a TATA box capable ofdirecting RNA polymerase II to initiate RNA synthesis at the appropriatetranscription initiation site for a particular coding sequence. Apromoter may additionally comprise other recognition sequences generallypositioned upstream or 5′ to the TATA box, referred to as upstreampromoter elements, which influence the transcription initiation rate. Itis recognized that having identified the nucleotide sequences for thepromoter regions disclosed herein, it is within the state of the art toisolate and identify further regulatory elements in the 5′ untranslatedregion upstream from the particular promoter regions identified herein.Thus the promoter regions disclosed herein are generally further definedby comprising upstream regulatory elements such as those responsible fortissue and temporal expression of the coding sequence, enhancers and thelike. In the same manner, the promoter elements which enable expressionin the desired tissue such as the seed can be identified, isolated, andused with other core promoters to confirm seed-preferred expression.

[0044] The regulatory sequences of the present invention, when operablylinked to a heterologous nucleotide sequence of interest and insertedinto a transformation vector, enable seed-preferred expression of theheterologous nucleotide sequence in the seeds of a plant stablytransformed with this vector.

[0045] By “seed-preferred” is intended expression in the seed, includingat least one of embryo, kernel, pericarp, endosperm, nucellus, aleurone,pedicel, and the like.

[0046] By “heterologous nucleotide sequence” is intended a sequence thatis not naturally occurring with the promoter sequence. While thisnucleotide sequence is heterologous to the promoter sequence, it may behomologous, or native, or heterologous, or foreign, to the plant host.

[0047] It is recognized that the promoters may be used with their nativecoding sequences to increase or decrease expression resulting in achange in phenotype in the transformed seed.

[0048] The isolated promoter sequences of the present invention can bemodified to provide for a range of expression levels of the heterologousnucleotide sequence. Thus, less than the entire promoter regions may beutilized and the ability to drive seed-preferred expression retained.However, it is recognized that expression levels of mRNA may bedecreased with deletions of portions of the promoter sequences.Generally, at least about 20 nucleotides of an isolated promotersequence will be used to drive expression of a nucleotide sequence.

[0049] It is recognized that to increase transcription levels enhancersmay be utilized in combination with the promoter regions of theinvention. Enhancers are nucleotide sequences that act to increase theexpression of a promoter region. Enhancers are known in the art andinclude the SV40 enhancer region, the 35S enhancer element, and thelike.

[0050] Modifications of the isolated promoter sequences of the presentinvention can provide for a range of expression of the heterologousnucleotide sequence. Thus, they may be modified to be weak promoters orstrong promoters. Generally, by “weak promoter” is intended a promoterthat drives expression of a coding sequence at a low level. By “lowlevel” is intended at levels of about 1/10,000 transcripts to about1/100,000 transcripts to about 1/500,000 transcripts. Conversely, astrong promoter drives expression of a coding sequence at a high level,or at about 1/10 transcripts to about 1/00 transcripts to about 1/1,000transcripts.

[0051] The nucleotide sequences for the promoters of the presentinvention may be the naturally occurring sequences or sequences havingsubstantial homology. By “substantial homology” is intended a sequenceexhibiting substantial functional and structural equivalence with thenaturally occurring sequence. Any structural differences betweensubstantially homologous sequences do not effect the ability of thesequence to function as a promoter as disclosed in the presentinvention. Thus, sequences having substantial sequence homology with thesequence of a particular seed-preferred promoter of the presentinvention will direct seed-preferred expression of an operably linkedheterologous nucleotide sequence. Two promoter nucleotide sequences areconsidered substantially homologous when they have at least about 70%,preferably at least about 80%, more preferably at least about 90%, stillmore preferably at least about 95% sequence homology. Substantiallyhomologous sequences of the present invention include variants of thedisclosed sequences such as those that result from site-directedmutagenesis, as well as synthetically derived sequences.

[0052] Substantially homologous sequences of the present invention alsorefer to those fragments of a particular promoter nucleotide sequencesdisclosed herein that operate to promote the seed-preferred expressionof an operably linked heterologous nucleotide sequence. These fragmentswill comprise at least about 20 contiguous nucleotides, preferably atleast about 50 contiguous nucleotides, more preferably at least about 75contiguous nucleotides, even more preferably at least about 100contiguous nucleotides of the particular promoter nucleotide sequencedisclosed herein. The nucleotides of such fragments will usuallycomprise the TATA recognition sequence of the particular promotersequence. Such fragments may be obtained by use of restriction enzymesto cleave the naturally occurring promoter nucleotide sequencesdisclosed herein; by synthesizing a nucleotide sequence from thenaturally occurring promoter DNA sequence; or may be obtained throughthe use of PCR technology. See particularly, Mullis et al. (1987)Methods Enzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology(Stockton Press, N.Y.). Again, variants of these promoter fragments,such as those resulting from site-directed mutagenesis, are encompassedby the compositions of the present invention.

[0053] Nucleotide sequences comprising at least about 40 contiguoussequences of the sequences set forth in SEQ ID NOS:1 and 4 areencompassed. These sequences may be isolated by hybridization, PCR, andthe like. Such sequences encompass fragments capable of drivingseed-preferred expression, fragments useful as probes to identifysimilar sequences, as well as elements responsible for temporal ortissue specificity. Biologically active variants of the promotersequences are also encompassed by the method of the present invention.Such variants should retain promoter activity, particularly the abilityto drive expression in seed or seed tissues. Biologically activevariants include, for example, the native promoter sequences of theinvention having one or more nucleotide substitutions, deletions orinsertions. Promoter activity may be measured by Northern blot analysis,reporter activity measurements when using transcriptional fusions, andthe like. See, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), herein incorporated by reference.

[0054] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “percentage ofsequence identity”, and (d) “substantial identity”.

[0055] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length promoter sequence, or the complete promotersequence.

[0056] (b) As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length and optionally can be30, 40, 50, 100, or more contiguous nucleotides in length. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

[0057] Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison may beconducted by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443; by computerized implementations of thesealgorithms, including, but not limited to: GAP, BESTFIT, BLAST, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup (GCG) (575 Science Drive, Madison, Wis.). An example of the BLASTfamily of programs, which can be used to search database sequencesimilarity for the purposes of this invention, includes BLASTN programfor nucleotide query sequences against nucleotide sequence dataset. See,Ausubel et al., eds. (1995) Current Protocols in Molecular Biology,Chapter 19 (Greene Publishing and Wiley-lnterscience, N.Y.).

[0058] The BLAST homology alignment algorithm is useful for comparingfragments of the reference nucleotide or amino acid sequence tosequences from public databases. It is then necessary to apply a methodof aligning the complete reference sequence against the complete publicsequence to establish a % identity (in the case of polynucleotides ) or% similarity (in the case of polypeptides). The GAP algorithm is such amethod.

[0059] GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol.48:443-453, 1970) to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater. Unlessotherwise stated, for purposes of the invention, the preferred method ofdetermining percent sequence identity is by the GAP version 10 algorithmusing default parameters.

[0060] GAP presents one member of the family of best alignments. Theremay be many members of this family, but no other member has a betterquality. GAP displays four figures of merit for alignments: Quality,Ratio, Identity, and Similarity. The Quality is the metric maximized inorder to align the sequences. Ratio is the quality divided by the numberof bases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff & Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

[0061] (c) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

[0062] (d) The term “substantial identity” of polynucleotide sequencesmeans that a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least 90%and most preferably at least 95%, compared to a reference sequence usingone of the alignment programs described using standard parameters.

[0063] Another indication that nucleotide sequences are substantiallyidentical is if two nucleic acid molecules hybridize to each other understringent conditions. Generally, stringent temperature conditions areselected to be about 5° C. to about 2° C. lower than the melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The denaturation or melting of DNA occurs over a narrow temperaturerange and represents the disruption of the double helix into itscomplementary single strands. The process usually is characterized bythe temperature of the midpoint of transition, T_(m), which is sometimesdescribed as the melting temperature. Formulas are available in the artfor the determination of melting temperatures. Typically, stringent washconditions are those in which the salt concentration is about 0.02 molarat pH 7 and the temperature is at 50, 55, or 60° C.

[0064] The nucleotide sequences for the seed-preferred promotersdisclosed in the present invention, as well as variants and fragmentsthereof, are useful in the genetic manipulation of any plant whenoperably linked with a heterologous nucleotide sequence whose expressionis to be controlled to achieve a desired phenotypic response. By“operably linked” is intended the transcription or translation of theheterologous nucleotide sequence is under the influence of the promotersequence. In this manner, the nucleotide sequences for the promoters ofthe invention may be provided in expression cassettes along withheterologous nucleotide sequences for expression in the plant ofinterest, more particularly in the seed of the plant.

[0065] Such expression cassettes will comprise a transcriptionalinitiation region comprising one of the promoter nucleotide sequences ofthe present invention, or variants or fragments thereof, operably linkedto the heterologous nucleotide sequence whose expression is to becontrolled by the seed-preferred promoters disclosed herein. Such anexpression cassette is provided with a plurality of restriction sitesfor insertion of the nucleotide sequence to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

[0066] The expression cassette will include in the 5′-to-3′ direction oftranscription, a transcriptional and translational initiation region, aheterologous nucleotide sequence of interest, and a transcriptional andtranslational termination region functional in plants. The terminationregion may be native with the transcriptional initiation regioncomprising one of the promoter nucleotide sequences of the presentinvention, may be native with the DNA sequence of interest, or may bederived from another source. Convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also, Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al.(1987) Nucleic Acid Res. 15:9627-9639.

[0067] The expression cassette comprising the promoter sequence of thepresent invention operably linked to a heterologous nucleotide sequencemay also contain at least one additional nucleotide sequence for a geneto be cotransformed into the organism. Alternatively, the additionalsequence(s) can be provided on another expression cassette.

[0068] Where appropriate, the heterologous nucleotide sequence whoseexpression is to be under the control of the promoter sequence of thepresent invention and any additional nucleotide sequence(s) may beoptimized for increased expression in the transformed plant. That is,these nucleotide sequences can be synthesized using plant preferredcodons for improved expression. Methods are available in the art forsynthesizing plant-preferred nucleotide sequences. See, for example,U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) NucleicAcids Res. 17:477-498, herein incorporated by reference.

[0069] Additional sequence modifications are known to enhance geneexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon-intron splice sitesignals, transposon-like repeats, and other such well-characterizedsequences that may be deleterious to gene expression. The G-C content ofthe heterologous nucleotide sequence may be adjusted to levels averagefor a given cellular host, as calculated by reference to known genesexpressed in the host cell. When possible, the sequence is modified toavoid predicted hairpin secondary mRNA structures.

[0070] The expression cassettes may additionally contain 5′ leadersequences in the expression cassette construct. Such leader sequencescan act to enhance translation. Translation leaders are known in the artand include: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Nat Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example,TEV leader (Tobacco Etch Virus) (Allison et al. (1986)); MDMV leader(Maize Dwarf Mosaic Virus) (Virology 154:9-20); human immunoglobulinheavy-chain binding protein (BiP) (Macejak et al. (1991) Nature353:90-94); untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625);tobacco mosaic virus leader (TMV) (Gallie et al. (1989) MolecularBiology of RNA, pages 237-256); and maize chlorotic mottle virus leader(MCMV) (Lommel et al. (1991) Virology 81:382-385). See also Della-Cioppaet al. (1987) Plant Physiology 84:965-968. Other methods known toenhance translation and/or mRNA stability can also be utilized, forexample, introns, and the like.

[0071] In those instances where it is desirable to have the expressedproduct of the heterologous nucleotide sequence directed to a particularorganelle, particularly the plastid, amyloplast or vacuole, or to theendoplasmic reticulum, or secreted at the cell's surface orextracellularly, the expression cassette may further comprise a codingsequence for a transit peptide. Such transit peptides are well known inthe art and include, but are not limited to, the transit peptide for theacyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase,and the like.

[0072] In preparing the expression cassette, the various DNA fragmentsmay be manipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, for example,transitions and transversions, may be involved.

[0073] Reporter genes or selectable marker genes may be included in theexpression cassette. Examples of suitable reporter genes known in theart can be found in, for example, Jefferson et al. (1991) in PlantMolecular Biology Manual, ed. Gelvin et al. (Kluwer AcademicPublishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737;Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.

[0074] Selectable marker genes for selection of transformed cells ortissues can include genes that confer antibiotic resistance orresistance to herbicides. Examples of suitable selectable marker genesinclude, but are not limited to, genes encoding resistance tochloramphenicol (Herrera Estrella et al. (1983) EMBO J. 2:987-992);methotrexate (Herrera Estrella et al. (1983) Nature 303:209-213; Meijeret al. (1991) Plant Mol. Biol. 16:807-820); hygromycin (Waldron et al.(1985) Plant Mol. Biol. 5:103-108; Zhijian et al. (1995) Plant Science108:219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet210:86-91); spectinomycin (Bretagne-Sagnard et al. (1996) TransgenicRes. 5:131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol7:171-176); sulfonamide (Guerineau et al. 1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker et al. (1988) Science 242:41 9423);glyphosate (Shaw et al. (1986) Science 233:478481); phosphinothricin(DeBlock et al. (1987) EMBO J. 6:2513-2518).

[0075] Other genes that could serve utility in the recovery oftransgenic events but might not be required in the final product wouldinclude, but are not limited to, examples such as GUS (b-glucoronidase;Jefferson (1987) Plant Mol. Biol. Rep. 5:387), GFP (green florescenceprotein; Chalfie et al. (1994) Science 263:802), luciferase (Riggs etal. (1987) Nucleic Acids Res. 15(19):8115 and Luehrsen et al. (1992)Methods Enzymol. 216:397-414) and the maize genes encoding foranthocyanin production (Ludwig et al. (1990) Science 247:449).

[0076] The expression cassette comprising the particular promotersequence of the present invention operably linked to a heterologousnucleotide sequence of interest can be used to transform any plant. Inthis manner, genetically modified plants, plant cells, plant tissue,seed, and the like can be obtained.

[0077] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat No.5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowskiet al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration(see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al.(1995) “Direct DNA Transfer into Intact Plant Cells via MicroprojectileBombardment,” in Plant Cell, Tissue, and Organ Culture: FundamentalMethods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabeet al. (1988) Biotechnology 6:923-926). Also see Weissinger et al.(1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) ParticulateScience and Technology 5:27-37 (onion); Christou et al. (1988) PlantPhysiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Dafta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

[0078] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that constitutive expression of the desired phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure constitutive expression of the desired phenotypiccharacteristic has been achieved.

[0079] The following examples are offered by way of illustration and notby way of limitation.

[0080] EXPERIMENTAL

[0081] Promoter regions for the maize genes end1 and end2 were isolatedfrom maize plants and cloned. These genes were selected as sources ofseed-preferred promoters based on the spatial expression of their geneproducts. The method for their isolation is described below.

EXAMPLE 1 Isolation of Promoter Sequences

[0082] The procedure for promoter isolation is described in the UserManual for the Genome Walker kit sold by Clontech Laboratories, Inc.,Palo Alto, Calif. Genomic DNA from maize line A63 was prepared bygrinding 10-day-old seedling leaves in liquid nitrogen, and the DNAprepared as described by Chen and Dellaporta (1994) in The MaizeHandbook, ed. Freeling and Walbot (Springer-Verlag, Berlin) with a fewminor modifications. Precipitated DNA was recovered using an inoculationloop and transferred to a 1.5 ml eppendorf tube containing 500 μl ofTE(10 mM Tris pH 8.0, 1 mM EDTA). The DNA was allowed to dissolve atroom temperature for 15 minutes, phenol extracted and 2-propanolprecipitated in 700 μl. The precipitate was recovered and washed with70% ethanol. The DNA was then placed in a clean 1.5 ml eppendorf tube toair dry and resuspended in 200 μl of TE. RNase A was added to 10 μg/mland the mixture was incubated at 37° C. for several hours. The DNA wasthen extracted once with phenol-chloroform, then chloroform, thenethanol precipitated and resuspended in TE. The DNA was then usedexactly as described in the Genome Walker User Manual (Clontech PT3042-1version PR68687). Briefly, the DNA was digested separately withrestriction enzymes Dral, EcoRV, Pvull, Scal, and Stul, all blunt-endcutters. The DNA was extracted with phenol, then chloroform, thenethanol precipitated. The Genome Walker adapters were ligated onto theends of the restricted DNA. The resulting DNA is referred to as DL1-DL5,respectively.

[0083] For isolation of specific promoter regions, two nonoverlappinggene-specific primers (27-30 bp in length) were designed from the 5′ endof the maize genes identified from sequence databases. The primers weredesigned to amplify the region upstream of the coding sequence, i.e. the5′ untranslated region and promoter of the chosen gene. The sequence ofthe primers are given below for each promoter described. The first roundof PCR was performed on each DNA sample (DL1-5) with Clontech primer AP1(sequence 5′-gtaatacgactcactatagggc-3′) and the gene-specific primer(gsp)1 with the sequences shown in SEQ ID NOS:2 and 5.

[0084] PCR was performed in a model PTC-100 thermal cycler withHotBonnet from MJ Research (Watertown, Mass.) using reagents suppliedwith the Genome Walker kit. The following cycle parameters were used: 7cycles of 94° C. for 2 seconds, then 72° C. for 3 minutes, followed by32 cycles of 94° C. for 2 seconds and 67° C. for 3 minutes. Finally, thesamples were held at 67° C. for 4 minutes and then at 4° C. untilfurther analysis.

[0085] As described in the User Manual, the DNA from the first round ofPCR was then diluted and used as a template in a second round of PCRusing the Clontech AP2 primer (sequence 5′-actatagggcacgcgtggt-3′) andgene-specific primer (gsp)2 with the sequences shown in SEQ ID NOS:3 and6.

[0086] The cycle parameters for the second round were: 5 cycles of 94°C. for 2 seconds, then 72° C. for 3 minutes. Finally, the samples wereheld at 67° C. for 4 minutes and then held at 4° C. Approximately 10 μlof each reaction were run on a 0.8% agarose gel, and bands (usually 500bp or larger) were excised, purified with the Sephaglas BandPrep kit(Pharmacia, Piscataway, N.J.) and cloned into the TA vector pCR2.1(Invitrogen, San Diego, Calif.). Clones were sequenced for verification.

EXAMPLE 2 Expression Data Using Promoter Sequences

[0087] Three promoter::GUS fusion constructs were prepared by themethods described below. All vectors were constructed using standardmolecular biology techniques (Sambrook et al., Supra). A reporter geneand a selectable marker gene for gene expression and selection wasinserted between the multiple cloning sites of the pBluescript cloningvector (Stratagene Inc., 11011 N. Torrey Pines Rd., La Jolla, Calif.).The reporter gene was the β-glucuronidase (GUS) gene (Jefferson, R. A.et al., 1986, Proc. Natl. Acad. Sci. (USA) 83:8447-8451) into whosecoding region was inserted the second intron from the potato ST-LS1 gene(Vancanneyt et al., Mol. Gen. Genet. 220:245-250,1990), to produceintron-GUS, in order to prevent expression of the gene in Agrobacterium(see Ohta, S. et al., 1990, Plant Cell Physiol. 31(6):805-813). Therespective promoter regions were ligated in frame to sites 5′ to the GUSgene. A fragment containing bases 2 to 310 from the terminator of thepotato proteinase inhibitor (pinII) gene (An et al., Plant Cell1:115-122,1989) was blunt-end ligated downstream of the GUS codingsequence, to create the GUS expression cassette. The 3′ end of theterminator carried a NotI restriction site.

[0088] The promoter fusion end1::GUS::pinII was constructed using theGUS::pinII plasmid digested with BamHI, filled in with Klenow, anddigested with NcoI. The promoter was isolated from the TOPOTA vector bydigestion with Aval, filled in with Klenow, and then digestion withNcol. The fragment was ligated into the digested expression cassette,and successful subcloning was confirmed by restriction digest andsequencing.

[0089] The Agrobacterium transformation plasmids were constructed byinserting the GUS expression cassette as a HindIII/NotI fragment and theBAR expression cassette as a NotI/SacI fragment between the right andleft T-DNA borders in pSB11 at HindIII and SacI sites. The GUS cassettewas inserted proximal to the right T-DNA border. The plasmid pSB11 wasobtained from Japan Tobacco Inc. (Tokyo, Japan). The construction ofpSB11 from pSB21 and the construction of pSB21 from starting vectors isdescribed by Komari et al. (1996, Plant J. 10:165-174). The T-DNA of theplasmids were integrated into the superbinary plasmid pSB1 (Saito etal., EP 672 752 A1) by homologous recombination between the twoplasmids. The plasmid pSB1 was also obtained from Japan Tobacco Inc. E.coli strain HB101 containing the expression cassettes was mated withAgrobacterium strain LBA4404 harboring pSB1 to create the cointegrateplasmid in Agrobacterium using the method of Ditta et al., (Proc. Natl.Acad. Sci. USA 77:7347-7351, 1980). Successful recombination wasverified by a SaII restriction digest of the plasmid.

EXAMPLE 3 Transformation and Regeneration of Maize Callus viaAgrobacterium

[0090] Preparation of Agrobacterium suspension:

[0091] Agrobacterium is streaked out from a −80° frozen aliquot onto aplate containing PHI-L medium and cultured at 28° C. in the dark for 3days. PHI-L media comprises 25 ml/l Stock Solution A, 25 ml/l StockSolution B, 450.9 ml/l Stock Solution C and spectinomycin (SigmaChemicals) added to a concentration of 50 mg/l in sterile ddH2O (stocksolution A: K2HPO4 60.0 g/l, NaH2PO4 20.0 g/l, adjust pH to 7.0 w/KOHand autoclave; stock solution B: NH4Cl 20.0 g/l, MgSO4.7H2O 6.0 g/l, KCl3.0 g/l, CaCl2 0.20 g/l, FeSO4.7H2O 50.0 mg/l, autoclave; stock solutionC: glucose 5.56 g/l, agar 16.67 g/l (#A-7049, Sigma Chemicals, St.Louis, Mo.) and autoclave).

[0092] The plate can be stored at 4° C. and used usually for about 1month. A single colony is picked from the master plate and streaked ontoa plate containing PHI-M medium [yeast extract (Difco) 5.0 g/l; peptone(Difco) 10.0 g/l; NaCl 5.0 g/l; agar (Difco) 15.0 g/l; pH 6.8,containing 50 mg/L spectinomycin] and incubated at 28° C. in the darkfor 2 days.

[0093] Five ml of either PHI-A, [CHU(N6) basal salts (Sigma C-1416) 4.0g/l, Eriksson's vitamin mix (1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl0.5 mg/l (Sigma); 2,4-dichlorophenoxyacetic acid (2,4-D, Sigma) 1.5mg/l; L-proline (Sigma) 0.69 g/l; sucrose (Mallinckrodt) 68.5 g/l;glucose (Mallinckrodt) 36.0 g/l; pH 5.2] for the PHI basic mediumsystem, or PHI-I [MS salts (GIBCO BRL) 4.3 g/l; nicotinic acid (Sigma)0.5 mg/l; pyridoxine.HCl (Sigma) 0.5 mg/l; thiamine.HCl 1.0 mg/l;myo-inositol (Sigma) 0.10 g/l; vitamin assay casamino acids (Difco Lab)1.0 g/l; 2, 4-D 1.5 mg/l; sucrose 68.50 g/l; glucose 36.0 g/l; adjust pHto 5.2 w/KOH and filter-sterilize] for the PHI combined medium systemand 5 ml of 100 mM (3′-5′-Dimethoxy-4′-hydroxyacetophenone, Aldrichchemicals) are added to a 14 ml Falcon tube in a hood. About 3 fullloops (5 mm loop size) Agrobacterium is collected from the plate andsuspended in the tube, then the tube is vortexed to make an evensuspension. One ml of the suspension is transferred to aspectrophotometer tube and the OD of the suspension adjusted to 0.72 at550 nm by adding either more Agrobacterium or more of the samesuspension medium, for an Agrobacterium concentration of approximately0.5×109 cfu/ml to 1×109 cfu/ml. The final Agrobacterium suspension isaliquoted into 2 ml microcentrifuge tubes, each containing 1 ml of thesuspension. The suspensions are then used as soon as possible.

[0094] Embryo isolation, infection and co-cultivation:

[0095] About 2 ml of the same medium (here PHI-A or PHI-I) used for theAgrobacterium suspension are added into a 2 ml microcentrifuge tube.Immature embryos are isolated from a sterilized ear with a sterilespatula (Baxter Scientific Products S1565) and dropped directly into themedium in the tube. A total of about 100 embryos are placed in the tube.The optimal size of the embryos is about 1.0-1.2 mm. The cap is thenclosed on the tube and the tube vortexed with a Vortex Mixer (BaxterScientific Products S8223-1) for 5 sec. at maximum speed. The medium isremoved and 2 ml of fresh medium are added and the vortexing repeated.All of the medium is drawn off and 1 ml of Agrobacterium suspension isadded to the embryos and the tube vortexed for 30 sec. The tube isallowed to stand for 5 min. in the hood. The suspension of Agrobacteriumand embryos was poured into a Petri plate containing either PHI-B medium[CHU(N6) basal salts (Sigma C-1416) 4.0 g/l; Eriksson's vitamin mix(100×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; silver nitrate 0.85 mg/l; gelrite (Sigma) 3.0 g/l;sucrose 30.0 g/l; acetosyringone 100 mM; pH 5.8], for the PHI basicmedium system, or PHI-J medium [MS Salts 4.3 g/l; nicotinic acid 0.50mg/l; pyridoxine HCl 0.50 mg/l; thiamine.HCl 1.0 mg/l; myo-inositol100.0 mg/l; 2, 4-D 1.5 mg/l; sucrose 20.0 g/l; glucose 10.0 g/l;L-proline 0.70 g/l; MES (Sigma) 0.50 g/l; 8.0 g/l agar (Sigma A-7049,purified) and 100 mM acetosyringone with a final pH of 5.8 for the PHIcombined medium system. Any embryos left in the tube are transferred tothe plate using a sterile spatula. The Agrobacterium suspension is drawnoff and the embryos placed axis side down on the media. The plate issealed with Parafilm tape or Pylon Vegetative Combine Tape (productnamed “E.G.CUT” and is available in 18 mm×50 m sections; Kyowa Ltd.,Japan) and incubated in the dark at 23-25° C. for about 3 days ofco-cultivation.

[0096] Resting, selection and regeneration steps:

[0097] For the resting step, all of the embryos are transferred to a newplate containing PHI-C medium [CHU(N6) basal salts (Sigma C-1416) 4.0g/l; Eriksson's vitamin mix (1000× Sigma-1511) 1.0 ml/l; thiamine.HCl0.5 mg/l; 2.4-D 1.5 mg/l; L-proline 0.69 g/l; sucrose 30.0 g/l; MESbuffer (Sigma) 0.5 g/l; agar (Sigma A-7049, purified) 8.0 g/l; silvernitrate 0.85 mg/l; carbenicillin 100 mg/l; pH 5.8]. The plate is sealedwith Parafilm or Pylon tape and incubated in the dark at 28° C. for 3-5days.

[0098] Longer co-cultivation periods may compensate for the absence of aresting step since the resting step, like the co-cultivation step,provides a period of time for the embryo to be cultured in the absenceof a selective agent. Those of ordinary skill in the art can readilytest combinations of co-cultivation and resting times to optimize orimprove the transformation frequency without undue experimentation.

[0099] For selection, all of the embryos are then transferred from thePHI-C medium to new plates containing PHI-D medium, as a selectionmedium, [CHU(N6) basal salts (SIGMA C-1416) 4.0 g/l; Eriksson's vitaminmix (1000×, Sigma-1511) 1.0 ml/l; thiamine.HCl 0.5 mg/l; 2.4-D 1.5 mg/l;L-proline 0.69 g/l; sucrose 30.0 g/l; MES buffer 0.5 g/l; agar (SigmaA-7049, purified) 8.0 g/l; silver nitrate 0.85 mg/l; carbenicillin (ICN,Costa Mesa, Calif.) 100 mg/l; bialaphos (Meiji Seika K. K., Tokyo,Japan) 1.5 mg/l for the first two weeks followed by 3 mg/l for theremainder of the time.; pH 5.8] putting about 20 embryos onto eachplate. The plates are sealed as described above and incubated in thedark at 28° C. for the first two weeks of selection. The embryos aretransferred to fresh selection medium at two-week intervals. The tissueis subcultured by transferring to fresh selection medium for a total ofabout 2 months. The herbicide-resistant calli are then “bulked up” bygrowing on the same medium for another two weeks until the diameter ofthe calli is about 1.5-2 cm.

[0100] For regeneration, the calli are then cultured on PHI-E medium [MSsalts 4.3 g/l; myo-inositol 0.1 g/l; nicotinic acid 0.5 mg/l,thiamine.HCl 0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l, Zeatin0.5 mg/l, sucrose 60.0 g/l, Agar (Sigma, A-7049) 8.0 g/l, Indoleaceticacid (IAA, Sigma) 1.0 mg/l, Abscisic acid (ABA, Sigma) 0.1 mM, Bialaphos3 mg/l, carbenicillin 100 mg/l adjusted to pH 5.6] in, the dark at 28°C. for 1-3 weeks to allow somatic embryos to mature. The calli are thencultured on PHI-F medium (MS salts 4.3 g/l; myo-inositol 0.1 g/l;Thiamine.HCl 0.1 mg/l, Pyridoxine.HCl 0.5 mg/l, Glycine 2.0 mg/l,nicotinic acid 0.5 mg/l; sucrose 40.0 g/l; gelrite 1.5 g/l; pH 5.6] at25° C. under a daylight schedule of 16 hrs. light (270 uE m-2sec-1) and8 hrs. dark until shoots and roots develop. Each small plantlet is thentransferred to a 25×150 mm tube containing PHI-F medium and grown underthe same conditions for approximately another week. The plants aretransplanted to pots with soil mixture in a greenhouse. GUS+events aredetermined at the callus stage or regenerated plant stage.

[0101] For Hi-II a preferred optimized protocol was 0.5×109 cfu/mlAgrobacterium a 3-5 day resting step and no AgNO3 in the infectionmedium (PHI-A medium).

EXAMPLE 4 In Situ Localization of End2 mRNA in Developing Maize Kernel

[0102] In situ hybridization was performed using the protocol ofJackson, D. P. (1991) In situ Hybridization in Plants, Molecular PlantPathology: A Practical Approach, D. J. Bowles, S. J. Gurr, and M.McPherson, eds. Oxford University Press, England, pp.63-74. Both a senseand antisense probe corresponding to a protein of the end2 cDNA wereused. Probes were labelled non-isotopically with digoxigenin andincubated with various sections of 5 DAP and 9 DAP (days afterpollination) kernels of maize line CJ27 which had been fixed andembedded. Following extensive washing to remove unbound probe, sectionswere incubated with anti-digoxigenin alkaline phosphatase to detectareas of probe hybridization. For end2, mRNA was detected specificallywith the antisense probe and restricted to the aleurone layer ofendosperm tissue. The sense control probe did not hybridize.

EXAMPLE 5 Northern Analysis of Gene Expression in Vegetative Tissue andDeveloping Kernels

[0103] Total RNA (10 μg) was size fractionated on a 1% formaldehydeagarose gel and transformed to a nitrocellular membrane. Membranes werehybridized under stringent conditions with ³²P-labelled probesrepresenting cDNA fragments of the various genes. After extensivewashing to remove unbound probe, membranes were exposed on X-ray film.RNA samples were obtained from vegetative tissues as well as)developingmaize kernels.

[0104] The end1 expression pattern showed no expression in vegetativetissues. End1 was predominantly expressed in early to mid-developmentwhole kernel. Expression was also seen in isolated 7 DAP (days afterpollination) embryo, 10 DAP endosperm and 10 DAP pericarp.

[0105] The expression pattern of end2 likewise showed no expression invegetative tissue. End2 showed expression in 9-14 DAP whole kernel. End2also showed expression in isolated 7 DAP embryo, 10 DAP endosperm, and10 DAP pericarp.

[0106] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0107] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 10 1 713 DNA Zea mays 1 ggctggtaaa aaccattatt aactttaaca tcgaatcaaaactgacaaat tttatacttt 60 cacagagcag cagaaattta tacaatatga ttgaatacaagatgtaggac ccgatggaga 120 gaattttttt gtctcctata tgcttgaata cccaacataatatcttcgca gcatactatc 180 tatctaatag aaaaattata atatagttaa atacttaagtagtatctagt ggatagaatt 240 caatatctca tacatgcatg aggagtaata tctactagacatgcaacata tttttatcta 300 tctaatagaa tatatataat aaagttaaat attatatgcatcacctacta tatataattt 360 gatatctttt agatgtataa gggactaaga ataatatctctagcacacat gcaatgcatt 420 atctatctaa atatattata taatagttaa atattaattatacgtagtct aaacctacat 480 ataagcctac ccatccccac ttagagatct cagtgtcacacatagaccat acatctcact 540 tcgccaagaa aatttcgtca acagttgaag ttatacccatggcaaaacta ctcttgggtt 600 tgctccttgc ccttgctatt ctagggacaa catcggctgctggttgtgta caagaagggc 660 gaattctgca gatatccatc acactggcgg ccgctcgagcatgcatctag agg 713 2 27 DNA Zea mays 2 tgttgggtat tcaagcatat aggagac 273 29 DNA Zea mays 3 ccatcgggtc ctacatcttg tattcaatc 29 4 1224 DNA Zeamays 4 tactataggg cacgcgtggt cgacggcccg ggctggtaaa aagtaattga acccaaaata60 tcatggtatg tttggtgaag acagtgatca gtgatttttt tatatctata tatatatcaa 120agatacttga ttttctagaa ggttcttttt gttgttttcc cttatgtttt tacgcatgat 180gcaattcttt ttgagaggtt tccgatgcat tgatgttatt gtattatctc ctatatatag 240gtcgacgtac attatgtatt gcaataacca gttaactgga tccagcttcg cttagttttt 300agtttttggc agaaaaaatg atcaatgttt cacaaaccaa atatttttat aacttttgat 360gaaagaagat caccacggtc atatctaggg gtggtaacaa attgcgatct aaatgtttct 420tcataaaaaa taaggcttct taataaattt tagttcaaaa taaatacgaa taaagtctga 480ttctaatctg attcgatcct taaattttat aatgcaaaat ttagagctca ttaccacctc 540tagtcatatg tctagtctga ggtatatcca aaaagccctt tctctaaatt ccacacccaa 600ctcagatgtt tgcaaataaa tactccgact ccaaaatgta ggtgaagtgc aactttctcc 660attttatatc aacatttgtt attttttgtt taacatttca cactcaaaac taattaataa 720aatacgtggt tgttgaacgt gcgcacatgt ctcccttaca ttatgttttt ttatttatgt 780attattgttg ttttcctccg aacaacttgt caacatatca tcattggtct ttaatattta 840tgaatatgga agcctagtta tttacacttg gctacacact agttgtagtt ttgccacttg 900tctaacatgc aactctagta gttttgccac ttgcctggca cgcgactcta gtattgacac 960ttgtatagca aataatgcca atacgacacc tggccttaca tgaaacatta tttttgacac 1020ttgtatacca tgcaacatta ccattgacat ttgtccatac acattatatc aaatatattg 1080agcgcatgtc acaaactcga tacaaagctg gatgaccctc cctcaccaca tctataaaaa 1140cccgagcgct actgtaaatc actcacaaca caacacatat cttttagtaa cctttcaata 1200ggcgtccccc aagaactagt aaac 1224 5 30 DNA Zea mays 5 acttatcaggctttggaggt cattctcaca 30 6 32 DNA Zea mays 6 tccatcagca tgagagagcctatggcaaac at 32 7 22 DNA Artificial Sequence primer 7 gtaatacgactcactatagg gc 22 8 19 DNA Artificial Sequence primer 8 actatagggcacgcgtggt 19 9 22 DNA Artificial Sequence commercial primer AP1 9gtaatacgac tcactatagg gc 22 10 19 DNA Artificial Sequence commericalprimer AP2 10 actatagggc acgcgtggt 19

What is claimed is:
 1. An isolated promoter that is capable of driving transcription in a seed-preferred manner, wherein the promoter comprises a nucleotide sequence selected from the group consisting of: a) a sequence comprising a fragment of the nucleotide sequence set forth in SEQ ID NO:4; and b) the nucleotide sequence set forth in SEQ ID NO:4.
 2. The isolated promoter of claim 1 that is capable of driving transcription in a seed-preferred manner, wherein the promoter comprises a fragment of the nucleotide sequence set forth in SEQ ID NO:4.
 3. The isolated promoter of claim 1 that is capable of driving transcription in a seed-preferred manner, wherein the promoter comprises a nucleotide sequence set forth in SEQ ID NO:4.
 4. An expression cassette comprising a promoter and a nucleotide sequence operably linked to the promoter, wherein the promoter is capable of initiating seed-preferred transcription of the nucleotide sequence in a plant cell, wherein the promoter comprises a nucleotide sequence selected from the group consisting of: a) a sequence comprising a fragment of the nucleotide sequence set forth in SEQ ID NO:4; and b) the nucleotide sequence set forth in SEQ ID NO:4.
 5. The expression cassette of claim 4 wherein the promoter comprises a fragment of the nucleotide sequence set forth in SEQ ID NO:4.
 6. The expression cassette of claim 4 wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO:4.
 7. A plant stably transformed with an expression cassette comprising a maize promoter and a nucleotide sequence operably linked to the promoter, wherein the promoter is capable of initiating seed-preferred transcription of the nucleotide sequence in a plant cell, wherein the promoter comprises a nucleotide sequence selected from the group consisting of: a) a sequence comprising a fragment of the nucleotide sequence set forth in SEQ ID NO:4; and b) the nucleotide sequence set forth in SEQ ID NO:4.
 8. The plant of claim 7 wherein the promoter comprises fragment of the nucleotide sequence set forth in SEQ ID NO:4.
 9. The plant of claim 7 wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO:4. 